2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2000 to August 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

This test was conducted according to the "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (July 13, 1974, Kanpogyo No.5, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.615, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 49 Kikyoku No.392, Basic Industries Bureau, Ministry of International Trade and Industry, Japan). This test method is essentially the same as that in the OECD Guideline for Testing of Chemicals "Ready Biodegradability: Modified MITI Test (I) (Guideline 301C, July 17, 1992)".

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Supplier: Solutia Japan Limited
Lot number: I2-20-99
Purity: >99%. The test substance was treated as 100% in purity.
Confirmation of test substance: Two infrared (IR) spectra of the test substance provided by the sponsor and the performing laboratory were confirmed to be identical.
Appearance: Colorless clear liquid
Storage condition: Cold storage place
Stability: The test substance was stable under the storage conditions, as shown by the
finding that IR spectra of the test substance before the start and after the termination
of the cultivation were identical.

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

On-site sludge sampling was carried out at the following 10 locations in Japan.
Fushikogawa city sewage plant (Sapporo-shi, Hokkaido)
Kashima industrial sewage plant (Kashima-gun, lbaragi)
Nakahama city sewage plant (Osaka-shi, Osaka)
Ochiai city sewage plant (Shinjuku-ku, Tokyo)
Kitakami River (Ishinomaki-shi, Miyagi)
Shinano River (Nishikanbara-gun, Niigata)
Yoshino River (Tokushima-shi, Tokushima)
Lake Biwa (Otsu-shi, Shiga)
Hiroshima Bay (Hiroshima-shi, Hiroshima)
Dookai Bay (Kitakyushu-shi, Fukuoka)
Sludge sampling
(1) City sewage
Return sludge from sewage plants were collected.
(2) Rivers, lake and sea
Surface water and surface soil which was in contact with the atmosphere were collected .

Preparation of activated sludge
Activated sludge was prepared as follows to maintain its uniformity. The filtrate ( 5 L) of the supernatant of the activated sludge cultivated about for 3 months was mixed with the mixed filtrate (5 L) of the supernatant of a sludge collected newly at each location. The mixed filtrate (10 L) was aerated after the pH value of the mixture was adjusted to 7.0±1.0.
Cultivation
Roughly 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Dechlorinated water was added to the remaining portion so that the total volume reached 10L. This mixture was aerated, and then a predetermined amount of synthetic sewage was added to the mixture so that the concentration of the synthetic sewage was 0.1 wt% in the volume of dechlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25±2 °C.
-Control and use
During cultivation, the appearance of the supernatant, sedimentation of the sludge, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked to maintain a normal state of sludge. It was confirmed that these were within the scope of the control standard stipulated in the "Testing Methods for New Chemical Substances". Micro flora in the activated sludge was microscopically observed and sludge with no abnormal symptoms is used for the test.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

Analysis by GC

Details on study design:

Preparations for test
(1) Measurement of concentration of suspended solid: The concentration of suspended solid was measured to determine the amount of activated sludge to add.
Method: In accordance with Japanese Industrial Standards (JIS) K 0102-1998-14.1
Date: May 22, 2000
Result: Concentration of suspended solid in the activated sludge was 4900 mg/L .
(2) Preparation of basal culture medium: Each 3 mL of solutions A, B, C and D were made up to 1000 mL with purified water and then the pH of this solution was adjusted to 7.0.
(3) Reference substance: Aniline (reagent grade, Showa Chemicals Inc. Lot No. HK-2732D) was used as a reference substance to confirm that the sludge is sufficiently active. The pass level was that the percentage biodegradation of aniline calculated from the BOD value after 7 and 14 days were 40% or over and 65% or over, respectively.

Preparation of test solutions
The following test solutions were prepared and cultured under the conditions described further below.
(1) Addition of test substance or aniline
(a) Test solution (water+ test substance) (n=1, Vessel No.4). In one test vessel, 30 mg of the substance supplied by the sponsor was accurately weighed and added to 300 mL of purified water, so that the concentration of the test substance reached 100 mg/L.
(b) Test solution (sludge+ test substance) (n=3, Vessel No.1, 2 and 3). In each test vessel, 30 mg of the substance supplied by the sponsor was accurately weighed and added to the basal culture medium [the volume was less than 300 mL by the volume of activated sludge (1.84 mL) inoculated] so that the concentration of the test substance reached 100 mg/L.
(c) Test solution (sludge+ aniline) (n=1, Vessel No.6). In one test vessel, 29.5 uL [30.0 mg = 29.5 uL x 1.022 g/cm^3 (density)] of aniline was added into the basal culture medium [the volume was less than 300 mL by the volume of activated sludge (1.84 mL) inoculated], so that the concentration reached 100 mg/L.
(d) Test solution (control blank) (n=1, Vessel No.5). In one test vessel, nothing was added to the basal culture medium [the volume was less than 300 mL by the volume of activated sludge (1.84 mL) inoculated].
(2) Inoculation of activated sludge
The activated sludge cultivated under the conditions described previously was added to each test vessel, (b), (c) and (d), so that the concentration of the suspended solid reached 30 mg/L.

Instruments and conditions of cultivation
(1) Instruments for cultivation: Closed system oxygen consumption measuring apparatus
(Coulometer : Ohkura Electric Co., Ltd.), (Data sampler : Asahi Techneion Co., Ltd.)
Vessel 300 mL in volume (improved type)
Absorbent for carbon dioxide: Soda lime No.1

(2) Conditions of cultivation
Cultivation temperature: 25±1 C
Cultivation duration: 28 days
Stirring method: Each test solution is stirred by a magnetic stirrer.

Observation and measurement of test conditions
(1) Observation of test solution: The appearance of the test solution was observed periodically and conditions of the instruments were checked properly.
(2) Measurement of biochemical oxygen demand (BOD): During the test period, the change in BOD of the test solutions was measured by autorecording using a data sampler. Cultivation temperature was measured and recorded once a day.

Analysis of test solution: After the termination of the cultivation, dissolved organic carbon and the test were
determined.
Pretreatment of test solutions for analysis: After the termination of the cultivation, the test solution (water+ test substance), the test solutions (sludge+ test substance) and the test solution (control blank) were pretreated for total organic carbon (TOC) analysis and gas chromatography ( GC) analysis.

Reference substance:

aniline

Key result

Parameter:

% degradation (O2 consumption)

Value:

89

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

Results with reference substance:

Percentage biodegradations of aniline calculated by the BOD values were 60 % and
69 % after 7 and 14 days, respectively. It was concluded that this test conditions were valid.

Calculation table for the percentage biodegradation by BOD
Vessel Contents	Day 7		Day 14		Day 21		Day 28		Mean Degradation (%)
	BOD (mg)	Deg. (%)	BOD (mg)	Deg. (%)	BOD (mg)	Deg. (%)	BOD (mg)	Deg. (%)
Sludge + Analine (pos. control.)	54.7	60	64	69	72.4	78	75.5	80
Control Blank (B)	0.6	--	1.3	--	2.3	--	2.9	--
Sludge + Test Substance #1	37.1	48	56.6	73	64	82	73.3	93	89
Sludge + Test Substance #2	36.7	48	56.2	73	65	83	69.9	89
Sludge + Test Substance #3	30.1	39	47.6	61	61.5	78	67.4	85
Water + Test Substance	0.8	--	0.9	--	1.4	--	14.9	--
Deg. = (BOD-B) / TOD x 100
TOD of test substance = 75.6 mg
TOD of analine = 90.3 mg
Calculation table for percentage biodegradation by G C
Sample Description	A	E	F	G	H
Standard solution 300 mg/L	647555
Water + Test Substance	n.d.	0	0
Sludge + Test Substance #1	n.d.	0	0	100	100
Sludge + Test Substance #2	n.d.	0	0	100
Sludge + Test Substance #3	n.d.	0	0	100
Control Blank (B)	n.d.
Amount of Test substance added: 30 mg
A = Peak area (uV.sec)
B = Final volumn: 300 mL
C = Ratio of portion used for analysis: 290/300
D = Recovery Rate:  Water + test substance: 94.7%, Sludge + test substance: 92.5%
E = Residual amount of test substance (mg):  E=I x (A/A(standard)) x (B/C) / (D/100) /1000
F = Percentage Residue (%)
G = Percentage biodegrdation (%):  G = [(30-E(sludge + test substance)) / 30] x 100
H = Average percent biodegrdation (%)
I = Concentration of standard solution:  300 mg/L

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Remarks:

The test substance achieved 89% biodegradation within 28 days as measured by BOD, and 100% biodegradation as measured by GC. Due to the frequency of measurements it cannot be determined conclusively if the 10-day window criterion was met, but due to the rapid and high degree of biodegradation, the substance is considered to be readily biodegradable.

Conclusions:

MCS-1562 was determined to be readily biodegradable but the 10-day window criterion was not specified in the final report. The first analyses of % biodegradation occurred on day seven where the test substance had achieved a mean of 45% degradation (> 10% degradation) and the next measurement occurred 7 days later at day 14 where the mean degradation of the test substance was 69% (>60%). Due to the frequency of measurements it cannot be determined conclusively if the 10-day window criterion was met, but due to the rapid and high degree of biodegradation, the substance is considered to be readily biodegradable.

Executive summary:

The biodegradation of MCS-1562 ( 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate) was evaluated following a test method similar to OECD 301C "Ready Biodegradability: Modified MITI TEST (I)", July 17, 1992. The test results showed the average percent biodegradation determined by BOD measurements to be 89%, while the percent biodegradation calculated using GC (gas chromatography) was 100%. The test substance is considered readily biodegradable.

Description of key information

The biodegradation of MCS-1562 ( 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate) was evaluated following a test method similar to OECD 301C "Ready Biodegradability: Modified MITI TEST (I)", July 17, 1992. The test results showed the average percent biodegradation determined by BOD measurements to be 89%, while the percent biodegradation calculated using GC (gas chromatography) was 100%. The test substance is readily biodegradable based on the % biodegradation for the treatment vessels (test material plus activated sludge) compared to the control vessels (reference substance plus activated sludge.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater